Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences.

Abstract

The present study was carried out with the objective of development of species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin. The cattle-specific LAMP primer set was designed by targeting mitochondrial D-loop gene. The conditions for LAMP reaction for amplification of template DNA from cattle using designed cattle-specific primer set were optimized for the components of mixture and temperature of reaction. Amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. The developed species-specific LAMP assay was evaluated for its specificity, sensitivity and validated in laboratory on samples from known, coded, binary meat admixture with other than cattle at relative percentage of 20%, 10%, 5% and 1%, Phire tissue direct PCR master mix treated tissues of cattle and on species-specific polymerase chain reaction assay positive samples. The developed LAMP assay using self-designed primer set was highly specific, amplifying the DNA template exclusively from cattle tissue under the optimized LAMP reaction conditions. The sensitivity assay using serially diluted DNA templates revealed lowest level of detection as 0.01ng of absolute DNA from target species. Laboratory validation substantiated the accuracy of assay in known/unknown (coded) samples and up to the 1% level of admixture in binary meat sample. DNA present in supernatant of Phire Animal tissue kit treated samples were also amplified successfully eliminating the extra step of extraction of genomic DNA. The developed assays exhibited comparable results with previously established species-specific PCR assay taken as gold standards. Thus, it was concluded that developed species-specific loop-mediated isothermal amplification assay was effective in identification of tissue of cattle origin.